Culture conditions that allow zymomonas mobilis to assimilate n2 gas as a nitrogen source during bio-ethanol production

ABSTRACT

Chemically defined culture medium and culture conditions that allow bacteria to assimilate dinitrogen gas (N 2 ) as a nitrogen source during bio-ethanol production are disclosed herein. Methods of bioethanol production using the chemically defined culture medium and culture conditions are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to International Application NumberPCT/US2015/067100, filed on 21 Dec. 2015, which claims priority to U.S.Provisional Patent Application No. 62/098,634, filed Dec. 31, 2014, thedisclosure of both of which is incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under DE-SC0008131awarded by the Department of Energy. The Government has certain rightsin the invention.

BACKGROUND OF THE DISCLOSURE

The field of the disclosure relates generally to growth conditions forbacterial bio-ethanol production. Particularly, bacteria are grown underanaerobic conditions in a medium that has little or no soluble nitrogencompounds (e g , ammonium), but supplemented with iron (Fe) andmolybdenum (Mo), under a N₂ headspace.

More ethanol is produced than any other bio-fuel in the world, withproduction rates nearly 4-times that of biodiesel. Most ethanol iscurrently produced from starch from food crops using fermentative yeastsuch as Saccharomyces cerevisiae. Ethanol derived from food crops can bereferred to as grain ethanol. Recently, there has been a surge inethanol production from non-food crops, known as cellulosic feedstocksor lignocellulosic feedstocks. Ethanol derived from cellulosicfeedstocks can be referred to as cellulosic ethanol. Cellulosic ethanoloffers more favorable land use than grain ethanol because the non-foodcrops do not have to compete for land against crops grown for food.Furthermore, cellulosic ethanol production offers lower greenhouse gasemissions as compared to the production of grain ethanol. Unfortunately,the price of cellulosic ethanol remains high relative to that ofgasoline. Efforts to lower the cost of cellulosic ethanol have primarilyfocused on the largest cost contributors, such as plant feedstocks andthe cellulases needed to break them down into usable sugars.

Further, cellulosic feedstocks have low nitrogen contents, and thus,have to be enriched with nitrogen supplements to allow theethanol-producing microbes to grow. These supplements further incur alarge cost for cellulosic ethanol plants. For example, cellulosicfeedstocks must be enriched with nitrogen supplements such as corn steepliquor (CSL), diammonium phosphate (DAP), ammonium hydroxide and/orother ammonium salts. A facility that produces over 50 MM gallons ofethanol per year has been estimated to incur CSL and diammoniumphosphate costs between $1.7-1.9 million per year. Other analysesestimated even higher CSL costs between $7.7-18.2 million per year.Furthermore, there are projections that the supply of CSL could notscale to support operations of billions of gallons of ethanol per year.Thus, a sustainable alternative nitrogen source is desired.

Nitrogen gas (N₂) is recognized as a sustainable source of nitrogen foragriculture as leguminous crops can be exploited for the symbioticrelationships they form with N₂-fixing bacteria. N₂-fixing bacteria areable to use N₂ gas as a nitrogen source via the enzyme, nitrogenase. N₂gas could also serve as an economical and environmentally benignnitrogen source for industrial fermentations. For example, pure N₂ couldbe produced on-site for $0.06-0.21 per 100 cubic feet of gas. However,like all yeast, the industrial ethanol-producing yeast are incapable ofusing N₂ as a nitrogen source. Thus, there is a need for anethanol-producing microbe that uses N₂ as a nitrogen source.

Based on the foregoing, there is a need in the art for alternativemethods for producing ethanol, as well as higher chain alcohols, such asbutanol. It would be further beneficial to reduce or eliminate the needfor added nitrogen supplements to ethanol-producing fermentations andother industrial bioprocesses such to provide additional cost savings.

BRIEF DESCRIPTION OF THE DISCLOSURE

It has now been found that Zymomonas mobilis (Z. mobilis) can be grownin anaerobic medium lacking soluble nitrogen compounds (e g , ammonium)and supplemented with iron (Fe) and molybdenum (Mo) under a N₂headspace. Further, these conditions allow for N2 fixation duringanaerobic fermentation via nitrogenase. Particularly, the N₂-fixingcapacity of Z. mobilis can eliminate the need for nitrogen supplements,such as CSL, DAP, and ammonium, thereby allowing for a more effective,cost-efficient bio-ethanol production process.

Accordingly, in one aspect, the present disclosure is directed to achemically defined culture medium comprising iron and molybdenum. In oneembodiment, the chemically defined culture medium comprises: Na₂HPO₄(5.75 mM), KH₂PO₄ (7 mM), NaCl (8.6 mM), calcium pantothenate (105 nM),MgSO₄ (1 mM), CaCl₂ (0.1 mM) and trace elements 0.1% (v/v), wherein thetrace elements comprise: nitrilotriacetic acid (20 g/L), MgSO₄(28.9g/L), CaCl₂.2H₂O (6.67 g/L), (NH₄)₆Mo₇O₂₄.4H₂O (18.5 mg/L),FeSO₄.7H₂O(198 mg/L), and Metals 44 (0.1% v/v). Metals 44 is comprisedof: ethylenediaminetetraacetic acid (2.5 μg/L), ZnSO₄.7H₂O (10.95 μg/L),FeSO₄.7H₂O (5 μg/L), MnSO₄.H₂O (1.54 μg/L), CuSO₄.5H₂O (0.392 μg/L),Co(NO₃)₂.6H₂O (0.25 μg/L), and Na₂B₄O₇.10H₂O (0.177 μg/L). The abovemedium may be supplemented with either NH₄Cl (10 mM) or N₂ gas as anitrogen source in sealed vessels under anaerobic conditions.

In another aspect, the present disclosure is directed to a method ofalcohol production. The method comprises: growing bio-fuel producingbacteria in a chemically defined culture medium in the presence of anitrogen source, wherein the chemically defined culture medium comprisesiron (Fe) and molybdenum (Mo) and wherein the nitrogen source comprisesN₂ gas.

In another aspect, the present disclosure is directed to a method ofalcohol production. The method comprising: growing bio-fuel producingbacteria in a hydrolysate of a cellulosic feedstock in the presence of anitrogen source wherein the nitrogen source comprises N₂ gas, with iron(Fe) and molybdenum (Mo).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts growth in miscanthus hydrolysate that simulates theavailable nitrogen in an industrial cellulosic hydrolysate when Ar(black) or N₂ (white) headspace was provided with various supplements asanalyzed in the Examples. The final cell density represents the finaloptical density (OD₆₆₀) when all glucose was consumed minus the initialOD₆₆₀. Error bars are s.d. (n=4). Te, trace elements, as used in thechemically defined medium: Fe, 0.71 mg/L FeSO₄.7H₂O; Mo, 0.026 mg/L(NH₄)₆Mo₇O₂₄.4H₂O; Pan, 105 nM calcium pantothenate.

FIGS. 2A & 2B depict iron and molybdenum concentrations that limitgrowth of N₂-fixing Zymomonas mobilis (ZM4) in miscanthus hydrolysatemedium.

FIGS. 3A & 3B depict utilization of N₂ gas by Z. mobilis (ZM4). (FIG.3A) Growth (gray squares), glucose consumption (black triangles), andethanol production (black circles) when provided NH₄ ⁺(closed symbols)or N₂ (open symbols) in an anaerobic chemically defined medium. Errorbars are s.d. (n=4). (FIG. 3B) Relative abundances for the M-57 fragmentof tert-butyl-dimethylsilyl-alanine (inset image; alanine atoms in bold)normalized to the most abundant ion. Alanine was obtained from acidhydrolysis of Z. mobilis protein. Gray, unlabeled standard; black, Z.mobilis grown with ¹⁵N₂+NH₄ ⁺; white, Z. mobilis grown with ¹⁵N₂. The10% residual abundance at m/z 260 (white) is due to unlabeled N₂ in thetest tubes (9 +/−5% of the N₂) and unlabeled N from the inoculum (0.9+/−0.1% of the cells). All 12 amino acids analyzed showed similardistributions (Table 2). Error bars are s.d. (n=3).

FIGS. 4A & 4B depict absolute net metabolic fluxes during growth in achemically defined medium with ¹³C-labeled glucose and either NH₄ ⁺(FIG.4A) or N₂ (FIG. 4B) determined using ¹³C-labeling patterns fromproteinaceous amino acids, measured extracellular fluxes, and biomasscomposition. The thickness of the arrow is proportional to the fluxcarried by a pathway or reaction. The thinnest arrows representreactions with a rate of 0.83 mmol ×g DCW⁻¹×h⁻¹. Values with standarddeviations are in Table 3.

FIGS. 5A & 5B depict the effect of the amount of supplied N₂ on Z.mobilis (ZM4) growth (A) and ethanol yield (B) in 10 mL batch culturesgrown in an anaerobic chemically defined medium with 17 mL of headspace.All cultures were laid horizontally and shaken at 225 rpm. Cultures wereincubated at 30° C. Error bars are s.d. (n=3-9). The volumes of N₂ gasadded were as follows: 0.1 mL=0.45 mM; 0.5 ml=2.2 mM; 1 ml=4.5 mM; 2ml=8.9 mM; 3 ml=13.4 mM; 17 ml=76.0 mM. The rest of the headspace gaswas argon.

FIGS. 6A & 6B depict the effect of temperature on Z. mobilis (ZM4)growth (A) and ethanol yield (B) in 10 mL batch cultures grown in ananaerobic chemically defined medium with a full N₂ headspace. Allcultures were laid horizontally and shaken at 225 rpm. Cultures wereincubated at 30° C. Error bars are s.d. (n=3). Only 12% of the glucosewas consumed in the samples at 40° C. at the time of sampling (98 h) dueto the low amount of growth. N.G., no growth; N.D., not determined.

FIGS. 7A & 7B depict the effect of initial pH on Z. mobilis (ZM4) growth(A) and ethanol yield (B) in 10 mL batch cultures grown in an anaerobicchemically defined medium with a full N₂ headspace. All cultures werelaid horizontally and shaken at 225 rpm. Cultures were incubated at 30°C. Error bars are s.d. (n=3). pH was adjusted using either 1M NaOH or 1MHCl with 1M NaCl to maintain osmolarity as follows: pH 3.6, 70 μLHCl+100 μL NaCl; pH 4.5, 65 μL HCl+100 μL NaCl; pH 6.1, 50 μL HCl+100 μLNaCl; pH 7, 100 μL NaCl; pH 8.2, 70 μL NaOH+30 μL NaCl; pH 9.2, 80 μLNaOH+10 NaCl; pH 11.1, 100 μL NaOH. Ethanol yield was not determined atpH 3.6 and 11.1 due to a lack of growth and/or glucose consumption.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure is generally related to culture medium and growthconditions that allow for N₂ fixation during anaerobic fermentation.Particularly, it has been found that, in one embodiment, a chemicallydefined culture medium can be prepared that allows bacteria to usenitrogenase to assimilate dinitrogen gas (N₂) as the sole nitrogensource during bio-ethanol production. It is believed that growing anethanol-producing microbe with the capacity to fix N₂ can provide for amore effective, cost-efficient bio-ethanol production process since N₂could be produced at an ethanol production facility for a fraction ofthe cost of typical industrial nitrogen supplements.

The present disclosure solves the problematic spending of millions ofdollars on industrial nitrogen supplements by providing a culture mediumand growth conditions that allow for N₂ fixation during anaerobicproduction of bio-fuels by bacteria, particularly, ethanol production byZymomonas mobilis. Z. mobilis is a bacterium that produces ethanol fromglucose at near-theoretical maximum yields. Z. mobilis produces slightlymore ethanol per glucose than yeast and 3-5-times faster on a per cellbasis. Z. mobilis also produces less residual biomass during ethanolproduction than yeast. Strains of Z. mobilis have been engineered thatcan also consume 5-carbon sugars such as xylose and arabinose. Theseengineered strains can therefore use sugars derived from both celluloseand hemicellulose. Z. mobilis has long been viewed as a potentialcompetitor to yeast. Recent advances in tools to genetically manipulateZ. mobilis and improve its tolerance to toxic byproducts formed duringthe hydrolysis of cellulosic feedstocks have positioned Z. mobilis to bean emerging competitor for ethanol production versus yeast.

Accordingly, in one embodiment, a chemically defined culture medium canbe prepared that allows bacteria to use nitrogenase to assimilatedinitrogen gas (N₂) as a nitrogen source during bio-ethanol production.In some embodiments, N₂ is the sole nitrogen source during bio-ethanolproduction. In other embodiments, N₂ may be used as the nitrogen sourceas a supplement to other nitrogen sources that are present in limitingamounts, such that N2 can be used when the other nitrogen sources areused up.

Generally, the chemically defined culture medium for use in the presentdisclosure may be a chemically defined minimal medium supplemented withiron (Fe) and molybdenum (Mo), which are the two metal cofactorsrequired for nitrogenase activity. As used herein, “chemically definedculture medium” refers to a growth medium in which all of the chemicalcomponents, as well as their exact concentrations, are known.

Particularly, the chemically defined culture medium of one embodiment ofthe present disclosure includes: Na₂HPO₄ (5.75 mM), KH₂PO₄(7 mM), NaCl(8.6 mM), and trace elements 0.1% (v/v). The trace elements solutioncontains: nitrilotriacetic acid (20 g/L), MgSO₄ (28.9 g/L), CaCl₂.2H₂O(6.67 g/L), (NH₄)₆Mo₇O₂₄.4H₂O (18.5 mg/L), FeSO₄.7H₂O (198 mg/L), Metals44 (0.1% v/v). Metals 44 is comprised of: ethylenediaminetetraaceticacid (2.5 g/L), ZnSO₄.7H₂O (10.95 g/L), FeSO₄.7H₂O (5 g/L), MnSO₄.H₂O(1.54 g/L), CuSO₄.5H₂O (0.392 g/L), Co(NO₃)₂.6H₂O (0.25 g/L),Na₂B₄O₇.10H₂O (0.177 g). After autoclaving, the following supplementswere added (final concentrations): calcium pantothenate (105 nM), MgSO₄(1 mM), and CaCl₂ (0.1 mM).

Alternatively, calcium pantothenate can be replaced by 0.01% v/v cornsteep liquor.

The chemically defined culture medium can further be supplemented witheither NH₄Cl (10 mM) or N₂ headspace as a nitrogen source. When NH₄Cl isnot provided, an equimolar concentration of NaCl is provided to maintainsimilar osmotic conditions, if desired.

In some particularly suitable embodiments, the culture medium issupplemented with 100% N₂ headspace as a nitrogen source. It should berecognized by one skilled in the art, however, that lower concentrations(e.g., less than 100%, less than 95%, less than 90% or lower) of N₂headspace may be used as long as O₂ gas is not present at concentrationsthat would prevent nitrogenase function. Particularly suitable N₂ gasconcentration ranges from about 0.45 mM (per liter of culture liquid) toabout 76 mM (per liter of culture liquid), including from about 13.4 mM(per liter of culture liquid) to about 76 mM (per liter of cultureliquid).

In an alternative embodiment, a reactor sparged with N₂ to supply thenitrogen source.

Alternatively, the culture medium can be a hydrolysate of a cellulosicfeedstock supplemented with iron and molybdenum under a N₂ headspace orsparged with N₂. Suitable cellulosic feedstock materials include anynon-food, cellulose rich material such as switchgrass, miscanthus, cornstover, poplar, rice straw, sorghum, wheat straw, wood chips, sawdust,newspaper, other wood and paper products, other agricultural residues,and the like, and combinations thereof. Cellulose can be released fromlignin and hemicellulose, and hemicellulose can be hydrolyzed to sugarmonomers using pretreatment methods such as acid hydrolysis, alkalinehydrolysis, liquid hot water, steam explosion, ammonia fiber explosionand the like, and combinations thereof. A secondary hydrolysis wouldfollow the pretreatment to release sugar monomers from the cellulose.Secondary hydrolysis would make use of enzymes, including cellulases.The molybdenum and iron minerals could be (NH₄)₆Mo₇O₂₄.4H₂O (0.0185mg/L) and FeSO₄.7H₂O (0.198 mg/L) or other molybdenum andiron-containing minerals.

In one embodiment of the disclosure, a hydrolysate medium derived frommiscanthus grass was found to contain sufficient pantothenate to supportfull growth of Z. mobilis but insufficient iron (Fe) and molybdenum (Mo)(see FIG. 1). The Fe and Mo concentrations that limit growth ofN₂-fixing Z. mobilis (ZM4) in miscanthus hydrolysate medium wereidentified (see FIG. 2). Particularly, miscanthus hydrolysate wasprepared as described in the Examples below with N₂ as the majornitrogen source and with three separate glucose supplements to simulatesugar that would be released by cellulases. For iron-limitationexperiments (FIG. 2A) the (NH₄)₆Mo₇O₂₄.4H₂O concentration was constantat 0.026 mg/L. For molybdenum-limitation experiments (FIG. 2B) theFeSO₄.7H₂O concentration was constant at 0.71 mg/L. The minimumconcentration of (NH₄)₆Mo₇O₂₄.4H₂O and FeSO₄.7H₂O to support full growthof Z. mobilis in miscanthus hydrolysate were identified as 0.026 mg/Land 0.355 mg/L, respectively (see FIG. 2).

Generally, the microorganisms for use with the culture medium can be anybio-fuel producing bacteria or archaea as known in the art. As referredto herein, “a bio-fuel producing microorganism”, “a bio-fuel producingbacterium”, and “bio-fuel producing archaeon” include microorganismsincluding bacteria and archaea that are capable of producing ethanol aswell as higher chain alcohols, such as propanol, butanol and biodiesels,through the fermentation of sugars or starches, or polysaccharides suchas cellulose, hemicellulose, or syngas. As referred to herein, “bio-fuelproducing bacteria” and “bio-fuel producing archaea” include bacteriaand archaea that can use N₂ as a nitrogen source. In one particularlysuitable embodiment, the bacterium is Zymomonas mobilis, particularly,Z. mobilis ZM4. In another embodiment, the bacterium is another strainof Z. mobilis . In yet another embodiment, the bacterium is anengineered strain of Z. mobilis that is capable of fermenting bothhexose and pentose sugars and shows higher resistance to toxic compoundssuch as acetic acid and furfurals. In another embodiment, the bacteriumis a bio-fuel-producing bacterium that is not of the genus Zymomonas.Such bacteria could include ethanol and butanol-producing bacteria, suchas Acetobacterium woodii, Ruminococcus albus, Fibrobacter succinogenes,and various Clostridia such as Clostridium phytofermentans, C.kluyverrii, C. pasteurianum, C. beijerinckii, C. autoethanogenum, C.ljungdahlii, C. carboxidivorans, and C. acetobutlylicum.

In another aspect, the present disclosure is directed to a method ofalcohol production. The method includes: growing a bio-fuel producingmicroorganism in a chemically defined culture medium in the presence ofa nitrogen source, wherein the chemically defined culture mediumcomprises iron and molybdenum and wherein the nitrogen source is N₂ gas.

Suitable bacteria bio-fuel producing microorganisms include bio-fuelproducing bacteria and bio-fuel producing archaea as described herein.

A particularly suitable N₂ gas of the method is provided as a N₂ gasheadspace. Particularly suitable N₂ gas concentration ranges from about0.45 mM (per liter of culture liquid) to about 76 mM (per liter ofculture liquid).

The chemically defined culture medium further includes: Na₂HPO₄, KH₂PO₄,and NaCl in a molar ratio of about 1:1.2:1.5. The chemically definedculture medium suitably includes Na₂HPO₄, KH₂PO₄, NaCl, and traceelements, wherein the trace elements comprise: nitrilotriacetic acid,MgSO₄, CaCl₂.2H₂O, (NH₄)₆Mo₇O₂₄.4H₂O, FeSO₄.7H₂O,ethylenediaminetetraacetic acid, ZnSO₄.7H₂O, FeSO₄.7H₂O, MnSO₄.H₂O,CuSO₄.5H₂O, Co(NO₃)₂.6H₂O, and Na₂B₄O₇.10H₂O, as described herein.

The method can further include autoclaving the chemically definedculture medium. The method can further include providing calciumpantothenate, MgSO₄, and CaCl₂ to the autoclaved chemically definedculture medium.

The alcohol produced can be propanol, ethanol and butanol.

The method can further include growing the bio-fuel producingmicroorganism at a temperature ranging from about 20° C. to about 40° C.

The method can further include growing the bio-fuel producingmicroorganism at a pH ranging from about 4 to about 9.2.

In another aspect, the present disclosure is directed to a method ofalcohol production, the method comprising: growing a bio-fuel producingmicroorganism in a hydrolysate of a cellulosic feedstock in the presenceof a nitrogen source, wherein the nitrogen source is N₂ gas.

Suitable bacteria bio-fuel producing microorganisms include bio-fuelproducing bacteria and bio-fuel producing archaea as described herein.

A particularly suitable N₂ gas of the method is provided as a N₂ gasheadspace. Particularly suitable N₂ gas concentration ranges from about0.45 mM to about 76 mM.

A particularly suitable hydrolysate of a cellulosic feedstock can be,for example, a hydrolysate of Miscanthus x giganteus.

The hydrolysate can further include iron and molybdenum.

The alcohol produced can be propanol, ethanol and butanol.

It has unexpectedly been found that using the culture medium andconditions of the present disclosure allow for fixation of N₂ bybio-fuel producing bacteria during anaerobic fermentation, and thisprocess results in decreased biomass, while high alcohol, andparticularly, ethanol, yield is maintained. It has further been foundthat the specific rate of alcohol production is faster during N₂fixation than when conventional nitrogen sources, such as ammonium, arepresent.

Various functions and advantages of these and other embodiments of thepresent disclosure will be more fully understood from the examplesdescribed below. The following examples are intended to illustrate thebenefits of the present disclosure, but do not exemplify the full scopeof the disclosure.

EXAMPLES Example 1

In this Example, the ability of Zymomonas mobilis (Z. mobilis) to growin chemically-defined minimal medium and fix N₂ was analyzed.

¹⁵N₂ was purchased from Cambridge Isotope Laboratories (Tewskbury,Mass.). All other chemicals were purchased from Sigma-Aldrich (St.Louis, Mo.) or Fisher Scientific (Waltham, Mass.).

Zymomonas mobilis (ZM4) was obtained from the ARS Culture Collection.Cultures were grown in 10-ml volumes in 27-ml anaerobic test tubes or in60-ml volumes in 160-ml serum vials at 30° C. Media were made anaerobicby bubbling with either N₂ or Ar and sealed with rubber stoppers andaluminum crimps as described in Gordon & McKinlay (Bacteriol.196:1231-1237 (2014)). Test tubes were laid flat and serum vials wereleft upright. All cultures were shaken at 225 rpm.

The chemically defined medium (ZYMM) contained: Na₂HPO₄ (5.75 mM),KH₂PO₄ (7 mM), NaCl (8.6 mM), and trace elements 0.1% (v/v). The traceelements solution contained: nitrilotriacetic acid (20 g/L), MgSO₄ (28.9g/L), CaCl₂.2H₂O (6.67 g/L), (NH₄)₆Mo₇O₂₄.4H₂O (18.5 mg/L), FeSO₄.7H₂O(198 mg/L), Metals 44 (0.1% v/v). Metals 44 is comprised of:ethylenediaminetetraacetic acid (2.5 g/L), ZnSO₄.7H₂O (10.95 g/L),FeSO₄.7H₂O (5 g/L), MnSO₄.H₂O (1.54 g/L), CuSO₄.5H₂O (0.392 g/L), Co(NO₃)₂.6H₂O (0.25 g/L), Na₂B₄O₇.10H₂O (0.177 g/L). After autoclaving,the following supplements were added (final concentrations): calciumpantothenate (105 nM), MgSO₄ (1 mM), and CaCl₂ (0.1 mM). ZYMM wassupplemented with either NH₄Cl (10 mM) or a 100% N₂ headspace as anitrogen source. When NH₄Cl was not provided, an equimolar concentrationof NaCl was provided to maintain similar osmotic conditions.

To introduce ¹⁵N₂ gas, a stir bar was inserted into the neck of thebreakseal flask that the ¹⁵N₂ was shipped in and then a sampling portwas attached to the neck. The sampling port consisted of a 5-mltuberculin syringe with the plunger replaced by a rubber stopper(Geo-Microbial Technologies, Ochelata, Okla.) that was connected to thebreakseal flask neck by rubber tubing. The stir bar was then used tobreak the seal. The headspace in anaerobic test tubes containinganaerobic media was evacuated with a vacuum pump. For each addition ofgas to an anaerobic test tube, the breakseal flask was over-pressurizedwith 15-m1 of Ar using a syringe to displace the ¹⁵N₂ into the samplingport. 15 ml of the resulting ¹⁵N₂/Ar mixture was then transferred to theevacuated test tube via syringe.

Z. mobilis was inoculated into anaerobic chemically defined mediumcontaining the metal cofactors for nitrogenase (i.e., Mo and Fe). Celldensity was assayed by optical density at 660 nm using a Genesys 20visible spectrophotometer (Thermo-Fisher). Dry cell weights weredetermined as described in McKinlay et al. (Metab. Eng. 9:177-192(2007)). Optical densities were converted into dry cell weights usingexperimentally determined conversion factors of 516 mg DCW/L/OD₆₆₀ forcultures grown with NH₄ ⁺and 423 mg DCW/L/OD₆₆₀ for cultures grown withN₂. Electron recoveries were calculated based on available electrons asdescribed in McKinlay & Harwood (Proc. Natl. Acad. Sci. U S A107:11669-11675 (2010)), assuming an elemental composition ofCH_(1.125)O_(0.531)N_(0.214) (MW: 24.625 g/mole) for Z. mobilis biomass.Isotopic enrichments (¹⁵N) in amino acids were determined by gaschromatography-mass spectrometry (Agilent) as described in McKinlay(Metab. Eng. 9:177-192 (2007)).

As shown in FIG. 3A, Z. mobilis grew with either NH₄ ⁺ or N₂. The growthrate with N₂ was about half that observed during growth with NH₄ ⁺(Table1). No growth was observed when NH₄ ⁺ and N₂ were omitted (Ar headspace)(data not shown). When Z mobilis was cultured with ¹⁵N₂, GC-MS analysisof amino acids from whole cell protein showed a mass shift of +1 (FIG.3B; Table 2). This assimilation of ¹⁵N₂ confirmed that Z. mobilis canfix N₂ and that growth was not due to contaminating nitrogen sources. Nomass shift was observed when NH₄ ⁺ and ¹⁵N₂ were provided together (FIG.3B).

TABLE 1 Comparison of growth and metabolic parameters during growth withNH₄ ⁺ vs. N₂. Growth Ethanol yield (g yield Sp. Ethanol Sp. Glucose DCW× (mole × Ethanol:Biomass productivity consumption Carbon ElectronNitrogen Growth mole mole (mmole × g (mmole × g rate (mmol × g RecoveryRecovery source rate (h⁻¹) glucose⁻¹) glucose⁻¹) DCW⁻¹) DCW⁻¹ × h⁻¹)CDW⁻¹ × h⁻¹) (%) (%) NH₄ ⁺ 0.35 ± 0.02 9.5 ± 0.2 1.88 ± 0.08 198 ± 4 68.5 ± 3.8  36.43 ± 0.16 100 ± 4 101 ± 4 N₂ 0.22 ± 0.01 4.3 ± 0.1 1.95 ±0.03 450 ± 17 99.5 ± 2.05 50.99 ± 0.11 100 ± 2 102 ± 2 Errors are s.d.(n = 4). DCW, dry cell weight.

TABLE 2 Normalized mass isotopomer distributions in proteinaceous aminoacids from ¹⁵N₂ labeling experiments. The amino group is always oncarbon 2. Values are normalized to the most abundant ion. Thedistributions shown have not been corrected for natural isotopicabundances. Amino acid Amino Fragment standard ¹⁵N₂ + NH₄ ⁺ ¹⁵N₂ acid(m/z) C m+ Ave sd Ave sd Ave sd Ala 232 2, 3 0 100 0 100 0 11 1 1 22 022 0 100 0 2 9 0 9 0 22 0 3 1 0 1 0 9 0 260 1-3 0 100 0 100 0 11 1 1 230 23 0 100 0 2 9 0 10 0 23 0 3 1 0 1 0 9 0 Gly 218 2 0 100 0 100 0 11 11 21 0 21 0 100 0 2 9 0 9 0 21 0 3 1 0 1 0 9 0 246 1, 2 0 100 0 100 0 121 1 22 0 22 0 100 0 2 9 0 9 0 22 0 3 1 0 1 0 9 0 Val 186 2-5 0 100 0 1000 10 2 1 17 0 17 0 100 0 2 5 0 5 0 17 0 3 1 0 2 0 6 0 4 0 0 0 0 1 0 2602-5 0 100 0 100 0 9 1 1 25 0 25 0 100 0 2 10 0 10 0 24 0 3 1 1 2 0 10 04 0 0 0 0 2 0 5 0 0 0 0 0 0 6 0 0 0 0 0 0 288 1-5 0 100 0 100 0 10 1 125 0 25 0 100 0 2 9 2 10 0 25 0 3 1 0 2 0 10 0 4 0 0 0 0 2 0 Leu 200 2-60 100 0 100 0 9 1 1 18 0 18 0 100 0 2 6 0 6 0 18 0 3 1 0 1 0 6 0 4 0 0 00 1 0 274 2-6 0 100 0 100 0 10 1 1 25 0 25 0 100 0 2 10 0 10 0 25 0 3 20 2 0 10 0 4 0 0 0 0 2 0 Ile 200 2-6 0 100 0 100 0 9 1 1 18 0 18 0 100 02 5 0 5 0 18 0 3 1 0 1 0 5 0 4 0 0 0 0 1 0 274 2-6 0 100 0 100 0 9 1 138 0 26 0 100 0 2 6 0 10 0 25 0 3 1 0 2 0 10 0 4 0 0 0 0 2 0 Met 218 2-50 100 0 100 0 8 2 1 17 0 17 0 100 0 2 9 0 9 0 17 0 3 1 0 1 0 9 0 4 0 0 00 1 0 292 2-5 0 100 0 100 0 10 1 1 25 0 25 0 100 0 2 14 0 14 0 25 0 3 30 3 0 14 0 4 1 0 3 1 2 0 Ser 288 2, 3 0 100 0 100 0 10 1 1 26 0 26 0 1000 2 10 0 10 0 26 0 3 2 0 2 0 10 0 4 0 0 0 0 2 0 302 1, 2 0 100 0 100 011 1 1 26 0 26 0 100 0 2 10 0 11 0 26 0 3 2 0 2 0 10 0 4 0 0 0 0 2 0 3622, 3 0 100 0 100 0 9 1 1 33 0 31 2 100 0 2 16 0 15 0 34 0 3 3 0 3 0 13 14 1 0 1 0 3 0 390 1-3 0 100 0 100 0 10 1 1 35 0 35 0 100 0 2 16 0 16 035 0 3 4 0 4 0 16 0 4 1 0 1 0 4 0 5 0 0 0 0 1 0 Thr 376 2-4 0 100 0 1000 9 1 1 34 0 35 0 100 0 2 16 0 16 0 34 0 3 4 0 4 0 16 0 4 1 0 1 0 3 0404 1-4 0 100 0 100 0 9 1 1 36 0 36 0 100 0 2 16 0 17 0 36 0 3 4 0 4 016 0 4 1 0 1 0 4 0 5 0 0 0 0 1 0 Phe 234 2-9 0 100 0 100 0 10 1 1 21 021 0 100 0 2 6 0 6 0 21 0 3 1 0 1 0 6 0 4 0 0 0 0 1 0 302 1, 2 0 100 0100 0 9 1 1 26 0 26 0 100 0 2 10 0 10 0 26 0 2 0 2 0 10 0 0 0 0 0 2 0336 1-9 0 100 0 100 0 10 1 1 30 0 30 0 100 0 2 11 0 11 0 29 0 3 2 0 2 011 0 4 0 0 0 0 2 0 Asp 302 1, 2 0 100 0 100 0 9 1 1 26 0 27 0 100 0 2 110 11 0 27 0 3 2 0 2 0 10 0 4 0 0 0 0 2 0 316 2-4 0 100 0 100 0 10 1 1 270 28 0 100 0 2 11 0 11 0 27 0 3 3 0 3 0 11 0 4 1 0 1 0 2 0 5 0 0 0 0 1 0390 2-4 0 100 0 100 0 9 1 1 34 0 35 0 100 0 2 16 0 16 0 35 0 3 4 0 4 016 0 4 1 0 1 0 4 0 5 0 0 0 0 1 0 418 1-4 0 100 0 100 0 9 1 1 36 0 36 0100 0 2 17 0 17 0 36 0 3 4 0 4 0 17 0 4 1 0 1 0 4 0 5 0 0 0 0 1 0 Glu330 2-5 0 100 0 100 0 9 1 1 28 0 29 0 100 0 2 11 0 11 0 29 0 3 2 0 2 011 0 4 0 0 0 0 2 0 404 2-5 0 100 0 100 0 9 1 1 36 0 36 0 100 0 2 17 0 170 36 0 3 4 0 4 0 16 0 4 1 0 1 0 4 0 5 0 0 0 0 1 0 432 1-5 0 100 0 100 09 1 1 37 0 37 0 100 0 2 17 0 17 0 37 0 3 4 0 4 0 17 0 4 1 0 1 0 4 0 5 00 0 0 1 0 Tyr 302 1, 2 0 100 0 100 0 10 1 1 27 1 27 0 100 0 2 11 0 10 027 0 3 2 0 2 0 10 0 4 1 0 1 0 2 0

Example 2

In this Example, analysis of ethanol yield, rate, and intermediarymetabolic fluxes from Z. mobilis (ZM4) fermentation during N₂-fixationin the chemically defined medium was conducted and compared tofermentation with ammonium.

Converting ½ N₂ into NH₄ ⁺ requires 4 electrons and 8 ATP, which couldimpact various metabolic parameters. Accordingly, substrates andproducts were quantified using a Shimadzu high performance liquidchromatograph (HPLC analysis) as described in McKinlay et al. (Appl.Environ. Microbiol. 71:6651-6656 (2005)).

HPLC analysis of ZM4 supernatants showed that the ethanol yield was thesame regardless of whether NH₄ ⁺ or N₂ was provided (Table 1 above).Thus, electrons were not diverted away from ethanol production tosupport nitrogenase activity. Instead, electrons were diverted away frombiosynthesis, as the growth yield with N₂ was 46% of that with NH₄⁺(Table 1). The ratio of ethanol to biomass during growth with N₂ wasover twice that observed with NH₄ ⁺(Table 1). Producing the same amountof ethanol with less residual biomass is a positive aspect forindustrial ethanol production.

Remarkably, the specific ethanol production rate and the specificglucose consumption rate (Table 1) were both 1.45-fold higher duringgrowth with N₂ versus NH₄ ⁺. This higher metabolic rate may efficientlysupply reducing power to both N₂ fixation and biosynthetic reactions.Growth with N₂ offers the industrial benefit of a higher specificethanol production rate.

To determine if N₂ fixation affected intermediary metabolic fluxes,¹³C-metabolic flux analysis was performed using two different isotopicmixtures. Cultures were grown in the defined medium with either 100%[1-¹³C] glucose in one experiment and a mixture of 80% unlabeled and 20%uniformly labeled glucose. Isotopic enrichments (¹³ C) in amino acidswere determined by gas chromatography-mass spectrometry (Agilent) asdescribed in McKinlay (Metab. Eng. 9:177-192 (2007)). Intermediarymetabolic fluxes were estimated from the glucose uptake rate,fermentation product ratios, the biomass composition for Z. mobilis ZM4(Lee et al. Microb. Cell Fact. 9:94 (2010)), and mass isotopomerdistributions from the parallel labeling experiments with the software,13CFLUX2 (Weitzel et al. Bioinformatics 29:143-5 (2013)) using theNAGNLP optimizer found in the NAGC library as described in McKinlay etal. (J. Biol. Chem. 289:1960-70 (2014)). Mass isotopomer distributionswere corrected for natural isotopic abundances using IsoCor software(Millard et al. Bioinformatics 28:1294-1296 (2012)). Data from parallellabeling experiments were fit to a single metabolic model as describedin Schwender et al. (J. Biol. Chem. 281:34040-34047 (2006)).

The resulting fluxes (normalized to glucose uptake rates) were similarregardless of whether N₂ or NH₄ ⁺ was provided (Table 3). The absolutecentral metabolic rates were higher during growth with N₂ compared togrowth with NH₄ ⁺, whereas biosynthetic rates were lower (see FIGS. 4Aand 4B, see also Table 3). The data describe a metabolic network thatrigidly dedicates flux to ethanol, but is flexible in its overallmetabolic rate (FIGS. 4A and 4B, see also Table 3).

TABLE 3 Net metabolic flux distributions and standard deviationsdetermined using ¹³C-labeling patterns. Normalized Flux Absolute flux(mole % of glucose uptake rate) (mmol × g DCW⁻¹ × h⁻¹) Reaction NH₄ ⁺ N₂NH₄ ⁺ N₂ Glucose → G6P  100 ± 0.38  100 ± 0.21 36.43 ± 0.14  50.99 ±0.11  Central metabolism G6P → Pyr + GAP 99.69 ± 0.38  99.85 ± 0.21 36.32 ± 0.14  50.91 ± 0.11  G6P → F6P 0.19 ± 0.01 0.09 ± 0.01 0.07 ±0.00 0.05 ± 0.01 GAP →3PG 99.41 ± 0.38  99.73 ± 0.21  36.22 ± 0.14 50.85 ± 0.11  3PG → PEP 97.85 ± 0.42  99.00 ± 0.23  35.65 ± 0.15  50.48± 0.12  PEP → Pyr 89.00 ± 0.24  94.54 ± 0.14  32.42 ± 0.09  48.21 ±0.07  F6P + GAP → E4P + R5P 0.14 ± 0.01 0.07 ± 0.01 0.05 ± 0.00 0.04 ±0.01 R5P + R5P → GAP + S7P 0.00 ± 0.04 0.07 ± 0.01 0.00 ± 0.01 0.04 ±0.01 Pyr → EtOH 180.74 ± 7.98  190.69 ± 3.48  65.84 ± 2.91  97.23 ±1.77  Cit → αKG + CO2 5.49 ± 0.35 2.81 ± 0.19 2.00 ± 0.13 1.43 ± 0.10OAA + AcCoA→ Cit 5.49 ± 0.35 2.81 ± 0.19 2.00 ± 0.13 1.43 ± 0.10 PEP +CO₂ → OAA 8.63 ± 0.33 4.39 ± 0.18 3.14 ± 0.12 2.24 ± 0.09 Pyr → AcCoA +C1 5.83 ± 0.4  2.86 ± 0.23 2.12 ± 0.15 1.46 ± 0.12 Biosyntheticreactions AcCoA→ 1.84 ± 0.19 0.85 ± 0.09 0.67 ± 0.07 0.43 ± 0.05 E4P →0.14 ± 0.01 0.07 ± 0.01 0.05 ± 0.00 0.04 ± 0.01 F6P → 0.05 ± 0.00 0.02 ±0.00 0.02 ± 0.00 0.01 ± 0.00 GAP → 0.13 ± 0.01 0.06 ± 0.01 0.05 ± 0.000.03 ± 0.01 G6P → 0.12 ± 0.01 0.06 ± 0.01 0.04 ± 0.00 0.03 ± 0.01 OAA →1.65 ± 0.16 0.77 ± 0.08 0.60 ± 0.06 0.39 ± 0.04 PEP → 0.22 ± 0.02 0.07 ±0.01 0.08 ± 0.01 0.04 ± 0.01 3PG→ 1.56 ± 0.15 0.72 ± 0.07 0.57 ± 0.050.37 ± 0.04 R5P → 0.14 ± 0.07 0.07 ± 0.03 0.05 ± 0.03 0.04 ± 0.02 αKG →5.49 ± 0.35 2.81 ± 0.19 2.00 ± 0.13 1.43 ± 0.10

Example 3

In this Example, it was determined whether N₂ fixation would occurduring growth on a cellulosic feedstock.

Z. mobilis (ZM4) was cultured in a dilute acid hydrolysate of Miscanthusx giganteus grass that had been grown without fertilizer. Cultures weregrown in 10-ml volumes in 27-ml anaerobic test tubes. Test tubes werelaid flat and shaken at 225 rpm. The hydrolysate simulated the amount ofnitrogen expected to be available in an industrial medium, but notnecessarily other industrial parameters.

Miscanthus hydrolysate medium was prepared as described in Sedlak & Ho(Appl. Biochem. Biotechnol. 113-116:403-16 (2004)) with somemodifications. Miscanthus x giganteus was grown without fertilizer atthe Energy Biosciences Institute, Urbana, Ill. It was harvested, dried,and chopped. Briefly, 100 grams was hydrolyzed in 1% H₂SO₄ for 1 hour at121° C. The hydrolysate was filtered and the pH adjusted to 10 withCaOH₂ and heated to 50° C. for 30 minutes. The hydrolysate was thencooled to room temperature and the pH was adjusted to 6 using H₃PO₄.Precipitate was removed by filtration with Whatman filter paper and theliquid was then filter sterilized using a 0.2 micron filter followed by1:1 dilution with water. Various supplements were added as indicated atthe following concentrations: trace elements (0.1% v/v),(NH₄)₆Mo₇O₂₄.4H₂O (0.026 mg/L), FeSO₄.7H₂O (0.71 mg/L), clarified cornsteep liquor (Sigma), and calcium pantothenate (105 nM). Corn steepliquor (CSL) was clarified by centrifugation at 16,000 ×g for 5 minutes.The diluted hydrolysate contained 25 mM of glucose and 56 mM of xylose.Additional glucose was added to raise the concentration to 71 mM. Twoadditional glucose supplements of 500 μmoles each were added during thefermentation to simulate the total amount of sugar expected ifcellulases were used to liberate additional glucose, and if a straincapable of xylose utilization, such as is disclosed in WO 1998050524A1,which is incorporated by reference to the extent it is consistentherewith, was used. Excess gas was expelled between each addition ofglucose to lower the pressure in the tubes as a safety precaution. Tubeswere flushed with either N₂ or Ar as appropriate after each addition ofglucose. Another set of tubes did not receive additional N₂ supplementsto test whether N₂ was limiting.

Cell density was assayed by optical density at 660 nm using a Genesys 20visible spectrophotometer (Thermo-Fisher). Substrate and products werequantified using a Shimadzu high performance liquid chromatograph asdescribed in McKinlay et al. (Appl. Environ. Microbiol. 71:6651-6656(2005)).

The lowest level of growth was observed when no nitrogen supplementswere provided (FIG. 1; Ar+Te). The addition of N₂ gas resulted in a1.4-fold higher cell density when trace elements were omitted (N₂), anda 2-fold higher cell density when trace elements were provided (N₂+Te).The Fe and Mo concentrations that limit growth of N₂-fixing Z. mobilis(ZM4) in miscanthus hydrolysate medium were identified (see FIG. 2).Particularly, miscanthus hydrolysate was prepared with N₂ as the majornitrogen source and with three separate glucose supplements to simulatesugar that would be released by cellulases. For iron-limitationexperiments (FIG. 2A) the (NH₄)₆Mo₇O₂.4.4H₂O concentration was constantat 0.026 mg/L. For molybdenum-limitation experiments (FIG. 2B) theFeSO₄.7H₂O concentration was constant at 0.71 mg/L. Mo and Fe, the twonitrogenase metal cofactors, were the only trace elements required toachieve this 2-fold higher cell level (N₂+Fe+Mo), and trace elementswere not limiting, since doubling their concentration did not affect thefinal cell density (N₂+2×Te) (FIG. 1). Separately, N₂ was not limiting,as final cell densities were the same with and without additional N₂supplements during culturing (data not shown). Likewise, the hydrolysatecontained sufficient essential pantothenate vitamin, since the additionof pantothenate did not affect the final growth level (FIG. 1). Thehighest level of growth with N₂ was comparable to that seen when 1% CSLwas provided. The ethanol yield from the ‘N₂+Fe+Mo’ condition was 97±2%of the theoretical maximum, which was slightly higher than the 94±1%observed with 1% CSL (t-test, P<0.05). The minimum concentration of(NH₄)₆Mo₇O₂₄.4H₂O and FeSO₄.7H₂O to support full growth of Z. mobilis inmiscanthus hydrolysate were identified as 0.026 mg/L and 0.355 mg/L,respectively (see FIG. 2). Thus, when provided with the necessarycofactors for nitrogenase activity, N₂ can substitute for CSL as anitrogen supplement under these simulated industrial conditions.

Example 4

In this Example, the ability of Zymomonas mobilis (Z. mobilis) to growand produce ethanol with different volumes of N₂ gas was determined.

10 mL batch cultures were grown in an anaerobic chemically definedmedium with 17 mL of headspace. All cultures were laid horizontally andshaken at 225 rpm. Cultures were incubated at 30° C.

As shown in FIG. 5A, the amount of N₂ provided limited the amount of Z.mobilis growth when supplied at an amount below 13.4 mM (mM of N₂ in thevessel per liter of culture liquid). The ethanol yield generated by Z.mobilis was not affected by the amount of N₂ supplied, as ethanol yieldsnear the theoretical maximum of 2 moles of ethanol per mole of glucoseconsumed were observed even when only 0.45 mM N₂ was supplied (FIG. 5B).

Example 5

In this Example, the effect of temperature on the ability of Zymomonasmobilis (Z. mobilis) to grow and produce ethanol was determined.

10 mL batch cultures were grown in an anaerobic chemically definedmedium with a full N₂ headspace at 22° C., 30° C., 37° C., 40° C. and42° C. All cultures were laid horizontally and shaken at 225 rpm. Only12% of the glucose was consumed in the samples at 40° C. at the time ofsampling (98 h) due to the low amount of growth.

As shown in FIG. 6A, Z. mobilis could grow with N₂ gas as the solenitrogen source up to a temperature of 40° C. At 37° C., the amount ofgrowth was about two-thirds that observed at 22° C. and 30° C. At 40°C., growth was severely limited, as was glucose consumption, within the98 h observation period (FIG. 6A). At all temperatures where growth wasobserved, Z. mobilis generated an ethanol yield near the theoreticalmaximum of 2 moles of ethanol per mole of glucose consumed (FIG. 6B).

Example 6

In this Example, the effect of pH on Zymomonas mobilis (Z. mobilis) togrow and produce ethanol was determined.

10 mL batch cultures were grown in an anaerobic chemically definedmedium with a full N₂ headspace. All cultures were laid horizontally andshaken at 225 rpm. Cultures were incubated at 30° C. pH was adjustedusing either 1M NaOH or 1M HCl with 1M NaCl to maintain osmolarity asfollows: pH 3.6, 70 μL HCl+100 μL NaCl; pH 4.5, 65 μL HCl +100 μL NaCl;pH 6.1, 50 μL HCl+100 μL NaCl; pH 7, 100 μL NaCl; pH 8.2, 70 μL NaOH+30μL NaCl; pH 9.2, 80 μL NaOH+10 NaCl; and pH 11.1, 100 μL NaOH. Ethanolyield was not determined at pH 3.6 and 11.1 due to a lack of growthand/or glucose consumption.

As shown in FIG. 7A, Z. mobilis grew with N₂ gas as the sole nitrogensource when the initial pH was between 4.5 and 9.2. The increase inoptical density observed at pH 11.1 was unlikely due to growth, sincenegligible glucose consumption and ethanol production were observed,suggesting that the increase in optical density was due to a factorother than growth, such as the precipitation of minerals. Final celldensities were notably lower outside of an initial pH range of 6.1 to8.2 (FIG. 7A). At all initial pH conditions where growth was observed,Z. mobilis generated an ethanol yield near the theoretical maximum of 2moles of ethanol per mole of glucose consumed (FIG. 7B).

The above results demonstrate the use of N₂ as an alternative nitrogensource for cellulosic ethanol production by Z. mobilis.

This written description uses examples to disclose the invention andalso to enable any person skilled in the art to practice the presentdisclosure, including making and using any mediums or systems andperforming any incorporated methods. The patentable scope of the presentdisclosure is defined by the claims, and may include other examples thatoccur to those skilled in the art. Such other examples are intended tobe within the scope of the claims if they have structural elements thatdo not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

What is claimed is:
 1. A chemically defined culture medium comprisingiron and molybdenum.
 2. The chemically defined culture medium of claim 1further comprising: Na₂HPO₄, KH₂PO₄, and NaCl in a molar ratio of about1:1.2:1.5.
 3. The chemically defined culture medium of claim 1comprising: Na₂HPO₄, KH₂PO₄, NaCl, and trace elements, wherein the traceelements comprise: nitrilotriacetic acid, MgSO₄, CaCl₂.2H₂O,(NH₄)₆Mo₇O₂₄.4H₂O, FeSO₄.7H₂O, ethylenediaminetetraacetic acid,ZnSO₄.7H₂O, FeSO₄.7H₂O, MnSO₄.H₂O, CuSO₄.5H₂O, Co(NO₃)₂.6H₂O, andNa₂B₄O₇.10H₂O.
 4. A method of alcohol production, the method comprising:growing a bio-fuel producing microorganism in a chemically definedculture medium in the presence of a nitrogen source, wherein thechemically defined culture medium comprises iron and molybdenum andwherein the nitrogen source comprises N₂ gas.
 5. The method of claim 4wherein the chemically defined culture medium further comprises:Na₂HPO₄, KH₂PO₄, and NaCl in a molar ratio of about 1:1.2:1.5.
 7. Themethod of claim 4 wherein the chemically defined culture mediumcomprises: Na₂HPO₄, KH₂PO₄, NaCl, and trace elements, wherein the traceelements comprise: nitrilotriacetic acid, MgSO₄, CaCl₂.2H₂O,(NH₄)₆Mo₇O₂₄.4H₂O, FeSO₄.7H₂O, ethylenediaminetetraacetic acid,ZnSO₄.7H₂O, FeSO₄.7H₂O, MnSO₄.H₂O, CuSO₄.5H₂O, Co(NO₃)₂.6H₂O, andNa₂B₄O₇.10H₂O.
 8. The method of claim 4 further comprising autoclavingthe chemically defined culture medium.
 9. The method of claim 8 furthercomprising providing calcium pantothenate, MgSO₄, and CaCl₂ to theautoclaved chemically defined culture medium.
 10. The method of claim 4wherein the N₂ gas comprises N₂ gas headspace.
 11. The method of claim10 wherein the N₂ gas ranges from about 0.45 mM of N₂ per liter ofculture medium to about 76 mM of N₂ per liter of culture medium.
 12. Themethod of claim 4 wherein the bio-fuel producing microorganism isselected from the group consisting of: Zymomonas mobilis, Acetobacteriumwoodii, Ruminococcus albus, Fibrobacter succinogenes, Clostridiumphytofermentans, Clostridium kluyverrii, Clostridium pasteurianum,Clostridium beijerinckii, Clostridium autoethanogenum, Clostridiumljungdahlii, Clostridium carboxidivorans, and Clostridiumacetobutlylicum.
 13. The method of claim 4 wherein the bio-fuelproducing microorganism is Zymomonas mobilis (ZM4).
 14. The method ofclaim 4 wherein the alcohol is selected from the group consisting ofpropanol, ethanol and butanol.
 15. A method of alcohol production, themethod comprising: growing a bio-fuel producing microorganism in ahydrolysate of a cellulosic feedstock in the presence of a nitrogensource, wherein the nitrogen source comprises N₂ gas.
 16. The method ofclaim 15 wherein the cellulosic feedstock is selected from the groupconsisting of switchgrass, miscanthus, corn stover, poplar, rice straw,sorghum, wheat straw, wood chips, sawdust, newspaper, other wood andpaper products, other agricultural residues, and the like, andcombinations thereof.
 17. The method of claim 15 wherein the N₂ gascomprises N₂ headspace.
 18. The method of claim 15 wherein the N₂ gasranges from about 0.45 mM of N₂ per liter of culture medium to about 76mM of N₂ per liter of culture medium
 19. The method of claim 15 whereinthe bio-fuel producing microorganism is selected from the groupconsisting of: Zymomonas mobilis, Acetobacterium woodii, Ruminococcusalbus, Fibrobacter succinogenes, Clostridium phytofermentans,Clostridium kluyverrii, Clostridium pasteurianum, Clostridiumbeijerinckii, Clostridium autoethanogenum, Clostridium ljungdahlii,Clostridium carboxidivorans, and Clostridium acetobutlylicum.
 20. Themethod of claim 15 wherein the bio-fuel producing microorganism isZymomonas mobilis (ZM4).